1--Field of the Invention
The present invention relates to improved clearing agents for use in targeting diagnostic or therapeutic agents to a target site in a mammal. The present invention also relates to improved methods for diagnosing or treating patients using improved clearing agents.
2--Description of Related Art
The detection of a target site benefits from a high signal-to-background ratio of detection agent. Therapy benefits from as high an absolute accretion of therapeutic agent at the target site as possible, as well as a reasonably long duration of uptake and binding. In order to improve the targeting ratio and amount of agent delivered to a target site, the use of targeting vectors comprising diagnostic or therapeutic agents conjugated to a targeting moiety for preferential localization has long been known.
Examples of targeting vectors include diagnostic or therapeutic agent conjugates of targeting moieties such as antibody or antibody fragments, cell- or tissue-specific peptides, and hormones and other receptor-binding molecules. For example, antibodies against different determinants associated with pathological and normal cells, as well as associated with pathogenic microorganisms, have been used for the detection and treatment of a wide variety of pathological conditions or lesions. In these methods., the targeting antibody is directly conjugated to an appropriate detecting or therapeutic agent as described, for example, in Hansen et al., U.S. Pat. No. 3,927,193 and Goldenberg, U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,460,459, 4,460,561, 4,624,846 and 4,818,709, the disclosures of all of which are incorporated herein by reference.
One problem encountered in direct targeting methods, i.e., in methods wherein the diagnostic or therapeutic agent (the "active agent") is conjugated directly to the targeting moiety, is that a relatively small fraction of the conjugate actually binds to the target site, while the majority of conjugate remains in circulation and compromises in one way or another the function of the targeted conjugate. In the case of a diagnostic conjugate, for example, a radioimmunoscintigraphic or magnetic resonance imaging conjugate, non-targeted conjugate which remains in circulation can increase background and decrease resolution. In the case of a therapeutic conjugate having a very toxic therapeutic agent, e.g., a radioisotope, drug or toxin, attached to a long-circulating targeting moiety such as an antibody, circulating conjugate can result in unacceptable toxicity to the host, such as marrow toxicity or systemic side effects.
Pretargeting methods have been developed to increase the target:background ratios of the detection or therapeutic agents. Examples of pre-targeting and biotin/avidin approaches are described, for example, in Goodwin et al., U.S. Pat. No. 4,863,713; Goodwin et al., J. Nucl. Med. 29:226, 1988; Hnatowich et al., J. Nucl. Med. 28:1294, 1987; Oehr et al., J. Nucl. Med. 29:728, 1988; Klibanov et al., J. Nucl. Med. 29:1951, 1988; Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos et al., J. Nucl. Med. 31:1791, 1990; Schechter et al., Int. J. Cancer 48:167, 1991; Paganelli et al., Cancer Res. 51:5960, 1991; Paganelli et al., Nucl. Med. Commun. 12:211, 1991; Stickney et al., Cancer Res. 51:6650, 1991; and Yuan et al., Cancer Res. 51:3119, 1991; all of which are incorporated by reference herein in their entirety.
In pretargeting methods, a primary targeting species (which is not bound to a diagnostic or therapeutic agent) comprising a first targeting moiety which binds to the target site and a binding site that is available for binding by a subsequently administered second targeting species is targeted to an in vivo target site. Once sufficient accretion of the primary targeting species is achieved, a second targeting species comprising a diagnostic or therapeutic agent and a second targeting moiety, which recognizes the available binding site of the primary targeting species, is administered.
An illustrative example of pretargeting methodology is the use of the biotin-avidin system to administer a cytotoxic radioantibody to a tumor. In a typical procedure, a monoclonal antibody targeted against a tumor-associated antigen is conjugated to avidin (or biotin) and administered to a patient who has a tumor recognized by the antibody. Then the therapeutic agent, e.g., a chelated radionuclide covalently bound to biotin (or avidin), is administered. The radionuclide, via its attached biotin (or avidin), is taken up by the antibody-avidin (or -biotin) conjugate pretargeted to the tumor.
Pretargeting is an approach which offers certain advantages over the use of direct targeting methods. For example, use of the pretargeting approach for the in vivo delivery of radionuclides to a target for therapy, e.g., radioimmunotherapy, reduces the marrow toxicity caused by prolonged circulation of a radioimmunoconjugate. This is because the radioisotope is delivered as a rapidly clearing, low molecular weight chelate rather than directly conjugated to a primary targeting molecule, which is often a long-circulating species.
One problem encountered with two-step pretargeting methods is that circulating primary targeting species (antibody-avidin or -biotin conjugate, for example) interferes with the targeting of active agent species (biotin- or avidin-active agent conjugate) at the target site by competing for the binding sites on the active agent-conjugate. This problem typically is avoided or minimized by the use of a three-step approach wherein a clearing agent is administered as an intermediate step of the above two-step approach. The clearing agent binds and removes circulating primary conjugate which is not bound at the target site.
Paganelli et al. (J. Nucl. Med. 31:735, 1990 and Cancer Res. 51:5960, 1991) disclose a 3-step approach wherein a biotinylated antibody is administered, followed by cold, i.e., non-labeled and non-conjugated, avidin to clear nontargeted antibody. Radiolabeled biotin is then administered which binds to the avidin retained in the body, presumably where the avidin has complexed to the biotinylated antibody.
When antibody-avidin is used as the primary targeting moiety, excess circulating conjugate is cleared by injecting a biotinylated polymer such as biotinylated human serum albumin. This type of agent forms a high molecular weight species with the circulating avidin-antibody conjugate which is quickly recognized by the hepatobiliary system and deposited primarily in the liver.
To speed up this hepatobiliary recognition process, the clearing agent may be substituted with sugar residues, primarily galactose, such that the galactosylated complex is recognized by the asialoglycoprotein receptors in the liver. By using a galactosylated biotin-protein, substantially all circulating streptavidin-antibody and galactosylated biotin-protein is deposited into the liver on the first pass through, making the clearing process very fast and efficient. With circulating avidin conjugate removed, excess biotin-chelate-radionuclide is rapidly eliminated, preferably renally. Because the radionuclide spends a very short time in circulation, considerably less marrow toxicity to the patient is seen compared to when the radionuclide is attached directly to the antibody.
Examples of this methodology are disclosed, e.g., in Axworthy et al., PCT Application No. WO 93/25240; Paganelli et al., "Monoclonal Antibody Pretargeting Techniques For Tumour Localization: The Avidin-Biotin System", Nucl. Med. Comm., Vol. 12:211-234, (1991); Oehr et al., "Streptavidin And Biotin As Potential Tumor Imaging Agents", J. Nucl. Med., Vol. 29:728-729, (1988); Kalofonos et al., "Imaging Of Tumor In Patients With Indium-111-Labeled Biotin And Streptavidin-Conjugated Antibodies: Preliminary Communication", J. Nucl. Med., Vol 31:1791-1796, (1990); Goodwin et al., "Pre-Targeted Immunoscintigraphy Of Murine Tumors With Indium-111-Labeled Bifunctional Haptens", J. Nucl. Med., Vol. 29:226-234, (1988). Improved pretargeting methods using the biotin-avidin system are disclosed, e.g., in U.S. Pat. Nos. 5,525,338, 5,736,119, 5,846,741 and 5,482,698 and co-pending U.S. patent applications Ser. Nos. 08,486,166, and 08/688,781, the disclosures of which are incorporated by reference herein in their entirety.
Pretargeting as it has been practiced to date suffers from certain drawbacks. First among these is the very low amount of radionuclide delivered to the target site compared to when the radionuclide is directly attached to an antibody. Using the above example, the problem of low target accretion is exacerbated by the choice of the clearing agent used for the avidin-antibody conjugate. In that approach, it has been found that the clearing agent also removes antibody-avidin conjugate previously bound to the target site. This substantially reduces the amount of antibody-avidin at the target, typically by 50% or more. Further, the use of a biotinylated clearing agent tends to partially block remaining antibody-avidin sites at the target, thereby reducing the number of sites available for binding by the biotinylated diagnostic or therapeutic agent.
A need exists, therefore, for improved clearing agents which work efficiently and rapidly, but do not block the binding sites at the target site, and which do not remove primary conjugate localized at the target site.